Determination of factors affecting gene regulation and/or gene replication

ABSTRACT

The invention relates to a method for determining a factor affecting a cell, which affects directly or indirectly the DNA, RNA and/or proteins of the cell or their synthesis machinery, in which recombinant DNA plasmid is transferred into the cell, and the initial point or points of the reproducing machinery responsible for its replication are subject to an adjustable promoter, which is controllable either by positive or negative feedback; the cell containing the recombinant DNA plasmid is brought into contact with the affecting factor; the affecting factor is allowed to affect the cell containing the recombinant DNA plasmid for a suitable time, after which the promoter adjusting the starting point of the reproducing machinery responsible for the replication of the recombinant DNA plasmid starts growing in the cell, unless the affecting factor has not inhibited the replication of the plasmid; the shift of the copy number of the recombinant DNA plasmid in the cell is determined directly or indirectly.

Biotests are methods where one uses living cells or organisms as toolsto detect different analytes. Many of those methods utilize bacterial oryeast cells. Procaryotic organisms and especially Escherichia colibacterium are very well characterized. Yeast cells are eucaryoticorganisms and grow as single cells. The cultivation of yeast is easierthan the cultivation of higher eucaryotes. Yeast cells grow in simplecultivation media and they do not need addition of complicated growthfactors. The knowledge of yeast is expanding rapidly and comprehensivemaps of genes are known. Hundreds of specific mutations for bothbacteria and yeast are known. With knowledge of specific mutations it ispossible to study the activity of specific reactions and metabolicpathways. For instance with antibiotic sensitive bacterial mutants traceamounts of antibiotics cause changes in the metabolism or in themembranes. Using antibiotic sensitive bacterial mutants, one is able todevelop very sensitive tests to measure residual antibiotics frombiological material. Bacteria and yeast with mutations in their DNArepair mechanisms, or mutants whose cell membranes might be porous fordifferent small molecular weight substances, e.g., antibiotics, are moresensitive to genotoxic substances than wild type. Using different mutantstrains, one is able to measure for the presence of antibiotics andtoxic or mutagenic agents. Genetic engineering techniques can be used totransfer new characteristics into bacteria or yeast cells. The newcharacteristics can be provided by proteins which are encoded byviruses. The protein do not exist naturally in the target organism. Useof genetic engineering techniques expands the applicability of bacteriaand yeast cells for use in biotests.

The universal genetic code of DNA is similar in each organism. Therelationship between carcinogenicity and mutagenicity is the basis forusing tests for mutagenic agents as prescreening tests for carcinogenicagents. Testing for carcinogenicity in animals is extremely expensiveand time consuming. Use of tests for mutagenic agents as a quickscreening method for carcinogenicity has raised hope and interest. Thequick screening method for carcinogenicity would decrease animal-basedcarcinogenicity testing.

The AMES-test (Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutat.Res. 31, 347) is the test used most often to screen for mutagenicagents. The AMES-test utilizes Salmonella typhimurium as a testorganism. Utilizing the AMES-test one is able to detect the genotoxicityof most mycotoxins, aromatic amines and polycyclic hydrocarbons.However, the Ames-test is not able to detect the genotoxicity ofcarcinogenic metal salts or chlorinated hydrocarbons. The S. typhimuriumstrains used in the AMES-test contain point mutations in thebiosynthetic route of the amino acid histidine. As the bacteria areexposed to the action of mutagenic substance, a reversion mutationoccurs in the gene for histidine biosynthesis and the bacterium startsto produce histidine endogenously. Endogenous production of histidinegives the cell the ability to grow on minimal growth medium containingno added histidine. A pitfall in the AMES-test is poor sensitivity andslow performance. In the AMES-test all other genotoxic changes such asthose acting on enzymes remain undetected. The test is also ratherexpensive for each particular compound tested.

A test for the detection of genotoxic substances based onbioluminescence is known (Ulizur, S., Weiser, I. and Yannai, S. (1980)Mut. Res., 74, 113-121). In this method, dark mutants of Photobacteriumleioanathi and P. fischeri are used. In the presence of genotoxicsubstances, these strains start to emit light. The theoreticalbackground of the method remains somewhat obscure. It has beenspeculated that the effect of removing a repressor or preventing itsformation combined with a change in the chromosomal DNA of the bacteriummight trigger the formation of light producing proteins. Differentgenotoxic substances act with different rates in this test due to thevariety of different classes of substances. This test is faster than theAmes-test but is by no means easier to use. The bacteria used in thetest should produce light during long and varying periods of time (30min to 10 h) depending on the substance. The bacteria used are notcapable of stably emitting light, which makes the method somewhatproblematic. Due to these facts, the method is not easily automated foruse in routine work when there are a lot of specimens to be analyzed.Additionally, being of marine origin, the cultivation temperature of thebacteria is rather low, 15° C.—it is not known how well the effect ofgenotoxic substances correlate to the effects on man whose bodytemperature is 37° C.

Antibiotics, used as medicines against microbial invasion, are detectedfrom body fluids in order to study the dosage and penetration of themedicine. The effective therapeutic range of the antibiotic is oftenrather narrow and the risks due to overdosage might be large. It is alsoimportant to measure the presence/concentration of antibiotics in meatand cow milk due to symptoms in people with allergies to antibiotics.The cow milk used in cheese production should not contain antibioticsdue to the fact that cheese making bacteria are not able to grow inantibiotic-contaminated milk. Common methods for detecting antimicrobialmedicines are microbiological methods performed on agar. A direct methodis to measure the inhibition of the growth of sensitive bacterialstrains. One can also measure some metabolic parameters, such as acidproduction of a sensitive strain of bacteria, using proper colorindicators.

Typical examples of agar diffusion tests are cylinder, hole or diskmethods. The difference between these tests is in the way the sample isapplied to the agar and also in the way the bacteria are utilized in thetest.

Since microbiological methods utilize bacteria or their spores, thesensitivity of the test bacteria is of utmost importance. In the testsdescribed above compromises had to be made in the choice of a suitabletest strain since great sensitivity against antimicrobial agents andother characteristics needed for the test strain have not been found inthe same strain of bacteria.

Major drawbacks when using microbes in antibiotic residue tests are slowspeed and insensitive performance. In these methods one controls thegrowth of a test strain and thus the test cannot be performed in anhour. This is due to the fact that growth of microbes is a slow processeven in its fastest mode. In addition, in many cases spores orfreeze-dried microbes are used which make the tests even slower toperform.

Antibiotic detection methods based on bioluminescence measurement areknown. Ulizur (1986, Methods Enzymol., 133, 275-284) describes threedifferent ways to use bioluminescence for the detection of antimicrobialagents: a) lysis-test, b) induction test and c) bacteriophage test. Inthe first one, the lux-genes isolated from Vibrio fischeri produceluciferase protein which in the presence of substrates produces light.The genes have been cloned into a plasmid and transferred to Bacillussubtilis. The B. subtilis strain utilized is sensitive to antibioticswhich affect bacterial membranes. Examples of such antibiotics arepenicillins and cephalosporins. In the lysis-test, thelux-gene-containing B. subtilis is grown together with a test sample. Ifthe test sample contains an antibiotic, the synthesis of cell wallcomponents is prevented and the bacteria are lysed. Thus the cultureyields lower light emission when compared to a culture lacking the testsample.

The induction test utilizes dim mutants of P. phosphoreum bacteria,which do not produce light. The induction test and other bioluminescencetests developed by Ulizur are based on exploitation of the chromosomalDNA of the target cell. Antibiotics affecting protein synthesis aredetected in the induction test. When the bacteria are incubated togetherwith compounds that bind to DNA, the bacteria start to produce light,i.e., protein synthesis is initiated. If there is any antibiotic presentaffecting the protein synthesis then there is a decline in lightemission. The amount of antibiotic present is quantitated when comparedto a blank without antibiotic. The induction test will not detectantibiotics that affect DNA synthesis and its basis is obscure. Toperform the induction test it is essential to add minimal salts such asCa²⁺ and Mg²⁺ ions which are known to diminish or completely prevent theaction of aminoglycosides (streptomycin, kanamycin, neomycin,erythromycin). In addition, the induction parameters are very strict. Ifsamples contain other antibiotics (for instance nalidixic acid) or othersubstances triggering light production there may be problems in theinterpretation of the results. The amount of bacteria in the test is acritical parameter. If the concentration of bacteria in the test is toohigh, the culture has to be aerated due to the absolute requirement ofoxygen for the bioluminescence reaction in these bacteria. Additionalproblems include a great number of potential inducers, special measuringdevices, and reproducibility of results.

The bacteriophage test can be utilized to detect antibiotics affectingDNA synthesis, transcription and translation. In this test, wild-type,light-emitting P. phosphoreum bacteria are infected with lyticbacteriophages. In the presence of an antibiotic, new infectious phagescannot be synthesized due to the fact that DNA-, RNA- or proteinsynthesis is blocked. In the presence of an antibiotic, the lightemission is unchanged compared to the initial light level. However, ifno antibiotic is present, the phages rapidly multiply and inactivate thehost bacteria thus making it incapable of producing light. Thebacteriophage test is difficult to perform since it is necessary to addphages (sometimes with different titers) to the assay mixture and theaddition of antibiotic has to be carefully timed. The bacteriophage testsuffers some of the same problems discussed with the induction test.Notably, the composition of assay mixture and the amount of bacteriaused in the assay.

The methods discussed above can be used to reveal the presence ofantibiotics generally but not to reveal individual antibiotics. Bychanging measuring conditions or by adding enzymes to degrade certaincompounds, one is able to block the effect of some antibiotics. There isa great demand for fast and simple methods to detect heavy metals,toxins, or food additives. At the moment detection of these compoundsmust be performed in central laboratories. The devices for detectingheavy metals, toxins, or food additives are extremely expensive and needspecially trained personnel to use them. Quick, qualitative testsperformed in the field could screen for those samples which need moresophisticated instrumentation and research. Thus, the pressure oncentral laboratories would diminish and determination of problematicsamples would be faster.

A commercial “Microtox” test is able to detect toxic substances fromenvironmental specimens. This test is based on the use of light emittingP. phosphoreum bacteria. A sample to be analyzed is incubated togetherwith bacteria and the presence of a toxic substance is evaluated from adecreased level of light produced by the bacteria when compared tocontrols. A severe drawback with this test is the need for high salinity(2%) by the organism. High salinity has been shown to decrease thebiological effect of heavy metals. In addition, an incubationtemperature of 15° C. can be an obstacle.

In addition to the Microtox test, several tests utilizing whole animalsor animal cell lines have been developed to measure toxic substances.Pitfalls in these methods include complicated cultivation of cells, slowperformance and a need for skilled personnel.

A test should be able to detect toxic and mutagenic substances fromdifferent waters such as from waste-, consuming-, raw- and groundwaterand from water for refreshment purposes. In addition, water needed forindustrial processes, food processes as well as raw water needed forpharmaceutical industry are of interest. The test should be able toevaluate samples from ground sediments and air for their toxicity andmutagenicity. The raw material used in food industry as well as qualitycontrol of food stuffs needs attention. From certain waters one shouldbe able to detect the organic material which could be used forrespiration and biosynthesis purposes by microbes contaminating thewater. The organic material can be simple sugars, organic acids,peptides or proteins, compounds containing amino or phosphate groupslinked to carbon chain etc. There is a need for rapid, non-expensivetests for these kind of compounds since the conventional methods takeseveral days to complete in order to be able to evaluate the quality ofwater used for various purposes.

The invention described here is based on known and accepted principlesof gene expression, the factors affecting gene regulation, and on theuse of recombinant-DNA in organisms such as bacteria and yeast.

Gene technology has made it possible to use bacteria and yeast cells ashosts to produce proteins that these organisms do not produce naturally.Several kinds of recombinant-DNA vectors have been prepared for thispurpose. Usually, the recombinant-DNA vectors are extrachromosomalplasmids. Recombinant-DNA plasmids can contain several genes and theycan replicate independently of host chromosomal DNA. Recombinant-DNAtechniques are utilized to transfer foreign genes into new host cells.The gene of interest can be cloned into a plasmid vector withrestriction enzymes and DNA ligase. Following cloning, therecombinant-DNA plasmid is transformed into a bacterial host usingmethods well known in the art. For expression in a eukaryotic cell, therecombinant-DNA plasmid usually contains DNA from bacteria, viruses, andeukaryotes. Recombinant-DNA is transferred into eukaryotic cells usingtechniques well known in the art, such as calcium-phosphateprecipitation or electroporation.

Many recombinant-DNA plasmids have been developed during the last fewyears where a gene of interest has been placed under control of a strongpromoter. A goal has been to create high expression of a foreign gene inan organism such as E. coli. In an E. coli bacterium, the production ofprotein from the gene of interest can account for as much as 25% of thetotal cellular protein (Caulcott & Rhodes, 1986, Trends in Biotech.,June, 142-146). Overexpression of a foreign gene can be deleterious tothe host cell or its metabolism. To overcome the potential problem ofoverexpression, plasmids with regulatable promoters were developed, andthe gene of interest is placed under the control of the regulatablepromoter. The production of protein from the gene of interest can beturned on when the microbes achieve an optimal growth phase. Cultivationof the microbes to the optimal growth phase is performed undernon-stressing conditions. Expression of the foreign gene is usuallycontrolled by adding a chemical to the medium which activates theregulatable promoter. For example, plasmid pCSS108 (Korpella & Karp,Biotechnol Lett., 10(6), 1988, 383-388), shown in FIG. 6, contains abacterial luciferase gene under the control of a regulatable promoter.Expression of bacterial luciferase is induced byisopropyl-β-D-thiogalactopyranoside (IPTG) added to the culture medium.In addition, physical parameters, such as an increase or decrease inincubation temperature might regulate protein production.

The plasmids commonly used contain one or more resistance determinants,with which to select from a large population of cells only those whichcontain the plasmid. The resistance determinants helps the cell tosurvive in circumstances which are poisonous to other cells. A selectionagent is added to the growth medium and cells which contain the plasmidare able to grow, but cells which lack the plasmid are not able to grow.The resistance determinant is a gene which encodes a protein thatdegrades or otherwise inactivates the poisonous factor. Examples of suchpoisonous factors are antibiotics which are present in the growthmedium. Several genes encoding resistance factors are known. Theresistance factor used most often is the gene encoding β-lactamase.β-lactamase degrades penicillins or β-lactams which are penicillinderivatives. As a result of β-lactamase enzyme activity, the poisonouscharacter of penicillin is lost and bacteria can grow. Other commonlyused resistance genes include those encoding chloramphenicolacetyltransferase, kanamycin acetyltransferase and tetrahydrofolatereductase.

Depending on the type of cells chosen, genes which carry the ability forthe cell to grow in the presence of tetracyclin, erythromycin,spectinomycin, streptomycin, sulfonamides, neomycin, thiostrepton,viomycin and colicins can also be used. Some resistance factors thateliminate or change the heavy metal present in the medium can also beused. Selection pressure in favor of a cell containing a plasmid canalso be achieved by transferring a gene encoding a function whichcomplements a growth defect in the organism. Normally, such growthdefects arise from a defective gene in the chromosome of the organism.These genes are normally those which encode for proteins participatingin an amino acid biosynthesis pathway. An essential amino acid isremoved from the growth medium and the cell cannot grow unless the aminoacid biosynthesis gene is present. The gene will be present in the cellif the plasmid is present. When the gene is expressed the amino acidbiosynthesis pathway will be complemented and the cell can grow. Theplasmids can also encode genes of other vital functions in the cell. Anexample of such genes include genes encoding proteins that participatein the formation of the cell wall.

The copy number of various plasmids inside the cell can vary from one toseveral hundred, to over a thousand. A plasmid often used, pBR322, has acopy number of about 60 whereas a derivative of it, pUC8, has a copynumber of about 500. A reason for the large difference between tworelated plasmids is due to a one base pair mutation in the origin ofreplication (ori) sequence of the plasmid (Chambers et al., 1988, GENE,68, 139-149). The copy number of a plasmid can be artificially increasedat a suitable phase of growth by constructing a vector where ori isplaced under the control of a strong and regulatable promoter. Atpresent several plasmids are known whose copy number can be artificiallyshifted up during the growth of microbes. These plasmids are mainly usedin industrial processes to produce foreign recombinant proteins in largequantities. Thus the use of these run-away replication vectors forpurposes described above does not rule out the possibility of using themin this invention for measuring different agents which affect the cell.As examples in this invention we describe different run-away plasmidswith which a change in copy number is possible. Run-away plasmidsstudied and used to produce foreign proteins belong to series pOU. Theorigin of replication region of pOU plasmids has been put under thecontrol of strong and regulatable P_(R) promoter of phage lambda (Larsenet al., 1984, GENE, 28, 45-54). The P_(R) promoter of phage lambda isregulated by a repressor protein, cI857, which is destroyed by heatingto 42° C. The protein can be produced from a lysogenic phage, i.e., aphage which is integrated in the chromosome of the host cell, from aplasmid where the coding sequence has been introduced or from anotherplasmid which belongs to a different incompatibility group. Plasmids ofdifferent incompatibility groups of are plasmids which are able toreplicate independently without the presence of another plasmid in thesame cell. When the repressor protein has been destroyed, P_(R) promoteris turned on and without control it starts to produce proteins calledcopB and repA (originating from low-copy number plasmid R1) as well astranscription products of these and the copA gene. These factors andespecially the overproduction of repA protein result in enhanced or evenuncontrolled production of the plasmid DNA in E. coli bacterium.

Yeast as well as bacteria are single cell organisms but yeast differfrom bacteria by being eucaryotic cells. Compared to higher eucaryoticcells yeast are far better characterized from the genetic point of view.The genetic maps of Saccharomyces cerevisiae and Schizosaccharomycesbombei are already known in great detail (Petes, 1980, Ann. Rev.Biochem., 49, 845-876). In addition, powerful methods to transfer genesinto yeast are known. Thus, yeast are commonly used hosts for rec-DNA.

Four types of rec-DNA vectors are used with yeast: integration plasmids(YIp), episomal vectors (YEp), replicating vectors (YRp), and artificialchromosomes. The integrating vectors of yeast can contain DNAoriginating from bacteria and part(s) of yeast genes. This type ofplasmid integrates exactly at certain point(s) in the yeast chromosome.The replicating yeast plasmids contain DNA from bacteria, part of yeastDNA and a specific area from yeast chromosome. The specific area fromthe yeast chromosome is responsible for the replication of the plasmid.

The specific area from the yeast chromosome permits the plasmid toreplicate as extrachromosomal DNA molecule in the yeast cell. Theepisomal plasmids contain DNA from bacteria, a yeast gene and a part orthe whole 2 micron plasmid of yeast (Hollenberg, 1982, Current Topics inMicrobiology and Immunology, 96, 119-144). Artificial chromosomes arelinear DNA vectors which are not well suited for expression ofheterologous proteins.

A plasmid for yeast whose copy number can be regulated has beendescribed. Centromeres are needed in yeast during the partition of achromosome in mitosis and meiosis phases. Centromeric DNA (CEN3) hasbeen extracted and transferred under the control of alcoholdehydrogenase promoter (ADH2). The ADH2 promoter is repressed byglucose. The action of a CEN3 plasmid can be controlled by the carbonsource used to cultivate yeast. When glucose is used as carbon sourcethe ADH2 promoter is repressed and CEN3 works normally by balancing theplasmid structure (YRp) during mitosis. If the carbon source in thegrowth medium is changed the plasmid starts to replicate and the copynumber can increase up to one hundred per yeast cell(Chlebowicz-Sledziewska, E. & Sledziewska, A., 1985, GENE, 39, 25-31).

The expression vectors used in yeasts normally contain the followingstrong regulatable promoters: alcohol dehydrogenase isoenzyme I (ADHI)gene promoter, phosphoglycerol kinase (PGK) promoter, repressible acidphosphatase (PHO5) promoter and the promoter for α-factor. ADHI is ayeast cytoplasmic enzyme. ADHI produces ethanol from acetaldehyde andneeds NADH as a cofactor. When yeast cells are cultivated in thepresence of glucose there is at least 1% ADHI protein from the totalamount of proteins in yeast. The PGK promoter can be controlled by thecarbon source (for example glucose) used. The expression of PHO5 can beprevented by the addition of inorganic phosphate and activated byeliminating inorganic phosphate from growth medium. The control of PHO5happens through a special regulation apparatus, which is formed fromPHO2, PHO4, PHO80 and PHO85 gene products (Bosfiana, K. A., 1980, Proc.Natl. Acad. Sci. USA, 77, 6541-6545). Some mutants (PHO4 and PHO80) areknown, which can be activated by a simple change in temperature. Thesemutant yeast cells grow at 35° C. and do not produce acid phosphataseenzyme even if inorganic phosphate is absent in the medium. If thecultivation temperature is shifted down, acidic phosphatase is producedefficiently whether there is phosphate or not in the medium. This systemto control the protein production by a change in the cultivationtemperature has been used to produce, for instance, interferons (Krameret al., 1984, Proc. Natl. Acad. Sci USA, 81, 367-370).

The use of higher eucaryotes as host cells for rec-DNA vectors toproduce foreign proteins is rapidly expanding. The goal of such use isto produce proteins of eucaryotic origin in large quantities. In anoptimal expression system it would be possible to produce proteins inseveral different types of cell lines. A fully regulatable expressionsystem for protein production would be an ideal solution. Theregulatable promoters used most often work only in certain host cellsystems. The regulation of these promoters is poor and the expressionvectors are based on DNA of tumor producing viruses, thus there alsoexists certain risks in their uses.

In higher eucaryotes gene expression can be regulated with the help ofthe following: simian virus (SV40) T-antigen, metallothionein genes,heat-shock genes, glucocorticoid hormones, DNA methylation or withanti-sense RNA. The antigen produced by SV40 controls its owntranscription. T-antigen is produced in large amounts immediately afterthe virus has infected the target cell. Later the T-antigen binds to itsown promoter and prevents transcription. If SV40-vectors are used forcloning, regulation of the T-antigen can be prevented by using asuitable temperature sensitive T-antigen mutant. In these casesT-antigen mutants produce T-antigen normally at high temperatures butthe production is prevented at room temperature (Rio et al., 1985,Science, 227, 23-28).

Metallothioneins are proteins which bind heavy metals. Many eucaryoticcells produce these proteins in the presence of heavy metals. It hasbeen estimated that there is an increase over fifty fold in theproduction of metallothioneins when cadmium is added to the growthmedium to a concentration of 4×10⁻⁶ molar (Hamer, D. H. & walling, M.J., 1982, J. Mol. Appl. Genet., 1, 273-288). The protein productioninduced by cadmium can be further increased by using low Cd²⁺-contentgrowth media.

Many promoters of heat-shock genes have been shown to be applicable andwell regulated in several different cell lines. The regulation of thesepromoters is performed simply by shifting the growth temperature. Thegenes are activated at high temperatures and produce proteins. At lowtemperatures the proteins are produced in low amounts or not at all. Thebest studied example is the heat-shock system of common fruit-fly,Drosophila melanogaster, in which a rise in temperature from 25° C. to37° C. causes the cessation of normal protein production and theheat-shock proteins start to emerge. A major heat-shock protein ishsp70. The regulation mechanisms of heat-shock protein expression arenot well known. By using heat-shock promoters (hsp70), it has beenpossible to increase the production of hGH (human growth hormone) up to1200-fold compared to unactivated cells (Dreano et al., 1985, GENE, 49,1-8).

The invention described here uses procaryotic and eucaryotic organisms,which have been carefully selected and which contain applicable rec-DNAvector constructions. By turning on the synthesis of DNA, RNA orproteins under strict control one is able to measure or detect eitherdirectly or indirectly all those factors which affect on the synthesismachineries described above. As the basis of measurement one can use theprotein product encoded by the rec-DNA vector, the marker protein or itsactivity or the overall metabolic activity. By activating thereplication of rec-DNA vector in a controlled fashion one is able tomeasure the amount of DNA formed directly by using radioactive labels orwith flow-cytometric techniques.

One is able to prepare suitable rec-DNA vectors for the measurement ofdifferent classes of chemicals depending on the target of the chemical.It is possible with the aid of E. coli bacteria containingrunaway-replication type plasmids to quantitate, for instance, compoundsinhibiting DNA synthesis (nucleotides) and DNA replication as well asthose compounds binding to DNA like several cancer drugs. Replication ofrunaway-replication plasmids is controlled, for instance, by aninducible promoter, P_(RE), of phage lambda. Thus, DNA synthesis and thereplication of a plasmid can be triggered at a predetermined point oftime. The analytes to be measured can be linked directly to thisregulatable and strong DNA biosynthesis which is not dependent on celldivision. If the synthesis or replication of DNA is inhibited the resultis seen in the copy number of plasmid. The copy number may be the sameor even decreased compared to the initial stage. In the uninhibitedcontrol cells the copy number of plasmid per cell may increase rapidly.The change in copy number can be measured either directly by measuringthe amount of DNA or indirectly by measuring the amount of gene productsor the activity encoded by the plasmid DNA. It is possible to determineagents that have very different mode of action on the cell with the aidof this kind of a plasmid. This is due to the fact that one can alsoengineer a gene encoding a marker protein under the control of a strongand regulatable promoter, the expression of which is measured in thetest. Thus everything affecting DNA, RNA, proteins, or theirbiosynthesis can be measured. If one wants to develop a broad range oftests which covers agents affecting cell wall, nucleic acids, proteinsand metabolism an ideal means of detecting these agents is based on thiskind of a runaway-replication plasmid. In these cases cells are allowedto replicate after which the promoter regulating the replication isactivated. Simultaneously, or after a certain period, the promoterregulating a gene encoding a marker protein is activated. A vector withsimilar characteristics can be developed for eucaryotic cells.

Another kind of approach is to use plasmids whose replication is tied tohost cell division. Rec-DNA multicopy plasmids in which the geneencoding the marker protein is under the control of a strong andregulatable promoter can be used to detect agents that affect cellmembranes, proteins and metabolism. Agents affecting DNA or cellmembranes can be detected with the system if actively dividing cells areused. The multicopy plasmid is synthesized for daughter cells and thesystem is sensitive to agents affecting DNA. Actively dividing cells arealso sensitive to agents affecting the cell membranes. If the strongpromoter regulating the expression of the marker protein is activatedthe system ill then also be sensitive to agents affecting the mRNA andprotein synthesis.

As a special application, when genes encoding luciferase are used, onecan determine agents that affect energy metabolism. This is due to thefact that the reactions catalyzed by luciferases use energy-richsubstances of cells. Agents that can affect the energetic state of thecell on all biosynthetic levels (replication, transcription andtranslation) or in metabolism, can be determined with the aid ofbioluminescence, i.e., formation of light emission by the cells. Aspecial case is bacterial luciferase, which uses central products ofmetabolism, NAD(P) and FMNH₂. Another special case is fire-fly and clickbeetle luciferases, which use a central metabolite, ATP, for lightproduction.

The nature of the invention described here makes it possible to use verydifferent kinds of measuring modes for example spectrophotometric,fluorometric, luminometric and visual methods. Spectrophotometricmethods can be an alternative when there is a gene cloned into plasmidwhose product can be measured by monitoring the change in color such asβ-galactosidase, alkaline phosphatase, amylases, peroxidases,glucuronidases or oxidoreductases. Fluorometric methods utilizefluorescent substrates developed for various enzymes, thus yieldingsomewhat greater sensitivity compared to spectrophotometric techniques.The luminometric method is performed with the aid of genes encodingeither bacterial or beetle luciferases. Several luminescent bacterialspecies exist such as V. harveyi, V. fischeri, P. leiognathi, P.phosphoreum, Xenorhabdus luminescens etc. Examples of luminescentbeetles are Luciola minarelica, Photinus pyralis, Pyrophorusplaaiothalamus etc. There several eucaryotic species in the sea whichluminesce, such as marine ostracod Vargula hilgendorfii, jellyfishAeguorea victoria, batrachoidid fish Porichtys notatus, pempherid fishParapriacanthus ransonneti etc., which could be useful in the future forvarious applications. Here an advantage over spectrophotometric andfluorometric measurement is the extremely sensitive detection of lightemission. An important benefit in luminescent methods is the possibilityto calibrate internally the measurements by using other intracellulargenes which encode luciferase emitting a different color which could bemeasured with a special two wavelength-detecting apparatus. The othergene can be cloned in the same rec-DNA vector, in another vectorbelonging to a different incompatibility group, inserted in the hostchromosome, or in a phage etc. An example is the click beetleluciferases, which emit four different colors. The wavelengths rangefrom 547 nm to 593 nm (Wood et al., 1989, Science, 244, 700-702). Theother gene resulting in different wavelength can be put under aninducible production system (indicator “gene”) or it can be expressedconstitutively (internal standard) to compensate possible secondaryeffects arising from heterologous samples. The use of a simple colorindicator is useful in cases where there is no need for high sensitivitybut where the simplicity and fast performance are more important. If thechanges in cell metabolism are to be detected one can use for exampletetrazolium salts which form a low-solubility formazan color whenreduced. In these cases genes encoding dehydrogenases or oxidoreductasesact as mediators of reducing quantities to yield the intense color offormazan. Also immunological methods (antibodies) coupled to sensitivemeasuring systems (RIA, FIA) are possible. Use of radioactive labels andflow cytometry in detecting the end point of the test are possible.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of a method based on a change inplasmid copy number.

FIG. 2 shows a schematic representation of possible effects differentagents will have on the cell and how the effects can be coupled to achange in plasmid copy number.

FIG. 3 shows a schematic representation of the efficiency of mRNA andprotein synthesis in a cell exposed to an agent inhibiting biosynthesis.

FIG. 4 shows biosynthetic routes which can be affected if proliferatingcells are utilized.

FIG. 5a shows construction of plasmid pCSS111.

FIG. 5b shows construction of plasmid pCSS123.

FIG. 5c shows genetic organization of plasmid pCSS302.

FIG. 5d shows genetic organization of plasmid pCSS305.

FIG. 6 shows construction of plasmid pCSS108.

FIG. 7a shows construction of plasmid pCSS112.

FIG. 7b shows genetic organization of plasmid pCSS301.

FIG. 7c shows construction of plasmid pCSS962.

FIG. 8 shows the quantity and quality of plasmid DNA extracted fromheat-treated E. coli cells incubated in the presence of nalidixic acid.

FIG. 9a shows the detection of nalidixic acid using E. coli cells clonedwith pCSS123.

FIG. 9b shows the detection of chloramphenicol using E. coli cellscloned with pCSS123.

FIG. 9c shows the detection of heavy metal, cadmium, using E. coli cellscloned with pCSS123.

FIG. 9d shows the detection of trimethoprim using E. coli cells clonedwith pCSS123.

FIG. 9e shows the detection of oflaxicin using E. coli cells cloned withpCSS123.

FIG. 9f shows the detection of UV light using E. coli cells cloned withpCSS123.

FIG. 9g shows the detection of oflaxacin using E. coli cells cloned withpCSS302.

FIG. 9h shows the detection of citroflaxicin using E. coli cells clonedwith pCSS302.

FIG. 9i shows the detection of citroflaxicin using E. coli cells clonedwith pCSS305.

FIG. 9j shows the detection of oflaxacin using E. coli cells cloned withpCSS305.

FIG. 10 shows a comparison of the detection of toxic substances in E.coli cells where protein synthesis is directed by the P_(L) promoter orthe lac promoter.

FIG. 11a shows the kinetics of light production for E. coli K-12 HItrp(c1857) cells cloned with pCSS112 and treated with chloramphenicol.

FIG. 11b shows the detection of ampicillin, oxytetracyclin, andstreptomycin using B. subtilis 1A40 cells cloned the luciferase genefrom click beetle (pCSS962).

FIG. 12 shows the detection of rifamicin

FIG. 13 shows the detection of oxytetracyclin.

FIG. 14a shows the detection of sulphite.

FIG. 14b shows the detection of cadmium, a heavy metal.

FIG. 15 shows the detection of rifampicin or oxytetracyclin using clickbeetle luciferase.

FIG. 16 shows the detection of chloramphenicol using β-galactosidase.

FIG. 17 shows the detection of glucose.

THE METHOD BASED ON THE CHANGE IN COPY NUMBER OF REC-DNA VECTOR

A cell builds its heritable material, DNA, from deoxyribonucleotides.Extrachromosomal or episomal DNA can exist as plasmids in the cell. Thereplication of the plasmids is not directly dependent on cellproliferation. Each plasmid has its own origin of replication toreplicate and divide into daughter cells in the course of cell division.However, the plasmid utilizes the host cellos DNA replication machineryfor its own replication.

FIG. 1 shows a schematic representation of a method based on the changein plasmid copy number. A cell contains a special plasmid (for examplerunaway-replication plasmid pCSS123) which can be induced to replicateat a predetermined point of time and in a controlled fashion. In thebeginning, the cell contains only few copies of the plasmid. The agentto be examined is allowed to affect the cell for a suitable period oftime, after which, the replication of the plasmid is initiated. Theplasmid will then replicate as much as possible in the presence of theagent. The replication of the plasmid can be triggered by adding acoupling chemical or by physical means, for example, shifting thetemperature high enough for replication to commence. Simultaneously orafter triggering the replication, the expression of the marker proteincan be turned on from the same special plasmid. In this case the degreeof the plasmid replication can be directly quantitated by measuring theamount of the marker protein or its activity which is dependent on thecopy number of the plasmid inside the cell. This has been described inFIG. 1 as an amount of enzyme activity produced by the plasmid encodedgene. This makes it possible to study and measure factors affecting thesynthesis of RNA, transcription, translation, cell walls, specificmetabolic pathways and enzyme activities.

Shown in FIG. 2, a schematic representation on the possible effectsdifferent agents will have on the cell and how the effects can becoupled to a change in plasmid copy number. The biosynthesis of DNA, RNAand protein are multistep processes and they need the cooperation ofseveral factors. Each step has both natural and artificial agents whichaffect the systems either by activating or inhibiting them. Forinstance, nalidixic acid has an effect on the replication of DNA byinhibiting the action of DNA polymerase.

Remarkably, the starting point is a few regulatable DNA molecules whichcan be induced to replicate without cell proliferation. This makes itpossible to use non-proliferating cells for the testing of effectors.Thus, the time used for the assay is not limited by the slow growth andproliferation of cells. The inventiveness of this method is based oncontrolled multiplication of the plasmid DNA and therefore on apossibility to investigate very large groups of compounds.

In the instant invention, advantage is taken of regulatable promotersand the machineries controlled by these promoters to increase the copynumber of a plasmid and/or production of a marker protein by the cell ata predetermined phase of growth. In this context, promoter means an areaof DNA where the enzyme RNA polymerase can bind, and where specialregulator proteins or other molecules can interact. Promoters cancontrol the expression of a gene beside the promoter or nearby thepromoter. Examples of inducible E. coli promoters include lac, trp,hybrid promoter tac, and lambda promoters P_(L) and P_(R). Thesepromoters differ in strength and in mode of induction. Promoters lac andtrp can be induced with chemicals whereas induction of P_(L) promotercan be induced by heat treatment.

Determination of Toxic Substances using a Method where ProteinBiosynthesis is Controlled by a Regulatable Promoter in rec-DNA Plasmid

In a cell, RNA, especially messenger RNA (mRNA), also containsinformation. Messenger RNA is synthesized from ribonucleotidetriphosphates. The ribonucleotide triphosphates are stored in the cell.Messenger RNA is synthesized according to each gene in the DNA. Specialtranscription machinery contains molecules responsible for thetranscription of DNA into RNA. Proteins are made according tomRNA-molecule templates using universally accepted principles. RNAsynthesis and the corresponding protein production encoded by the mRNAcan be switched on very quickly. Special rec-DNA plasmids are used inthe instant invention. The special rec-DNA plasmids have been preparedso they can be activated and produce large amounts of selected proteins.For this purpose, a special plasmid has been constructed so the plasmidcopy number is constant in certain host cells. The plasmid copy numbercannot be selected in other host cells. The plasmid used should be highcopy number. High copy number plasmids correspond to high proteinproduction as more copies of the gene are present. As cells containingthe special rec-DNA plasmids are treated with agents prior to induction,the amount of agent, mode of action, or overall presence in the systemcan be determined by measuring protein production and determining ifprotein production is altered. Agents include, for example, antibioticswhich affect mRNA synthesis or antibiotics which affect proteinsynthesis. Utilizing the method described herein, agents can beidentified which specifically affect and control biosynthetic routes.Using rec-DNA plasmids as described herein, agents can be identified onwhich selected protein production is dependent.

Remarkably, agents which affect DNA synthesis and cell membranes can beidentified with the methods described herein. This is possibly due tothe fact that when a microbe is proliferating it is forced to synthesizethese high-copy number plasmids and hence these special plasmids aresusceptible to agents which affect DNA synthesis. Actively proliferatingcells are especially susceptible to agents that act against cellmembranes and their biosynthesis.

FIG. 3 shows the practical performance of the invention described abovein a case where the special plasmid exists at high copy number in themicrobe. The microbe was exposed to an agent inhibiting biosynthesis andafter a period of time the special plasmids were activated to produceprotein. In this example the protein is an enzyme. The thickness of thearrows shown in FIG. 3 correspond to the efficiency of mRNA and proteinsynthesis, and thus also to the efficiency of the agents. When theinhibiting agent was present, production of the enzyme remained low.This contrasts with the case where the affecting agent was not presentor there were known amounts of the agent.

FIG. 4 shows those biosynthetic routes which can be affected with therec-DNA plasmid described above if proliferating cells are used. If oneuses actively proliferating cells in the measurement, it is alsopossible to study agents affecting DNA as is shown in FIG. 2. Thedifference from FIG. 1 is the initial amount of plasmid copies in thecell and the possibility of artificially elevating the copy number innon-proliferating cells.

Promoters exist which can be induced by suitable treatments. Thestrength of promoters varies and the promoters described in thisinvention are rather strong. However, the P_(L) promoter is strongerthan the lac or trp promoters. The P_(L) promoter of phage lambda isalso much faster, i.e., the effect of induction is clearly seen muchearlier when compared to the lac or trp promoters. The slow inductionrate of the lac and trp promoters can be partly explained by the slowuptake of the inducing molecule through the cell membranes. Further, therate of induction is affected by the rate at which the inducer transitsthe inside of the cell to its effector site. In addition, the copynumber of the plasmids also causes relative differences in the inductionrate. The copy number of plasmid pCSS112 (see FIG. 7) in E. coli isabout 60, whereas plasmid pCSS108 (see FIG. 6) has a copy number ofaround 600. The production of bacterial luciferase by the plasmids iscontrolled in pCSS112 by the P_(L) promoter of phage lambda and inpCSS108 by the lac promoter of E. coli lactose operon. Both promotersare controlled by certain repressor proteins, which are produced inlimited amounts. As the copy number of plasmid in the former case is tentimes lower than in the latter case, the production of luciferaseprotein is better shut down, i.e., repressed. In the latter case, thelac promoter leaks due to the relatively low amount of repressor proteinand thus the basal level of the luciferase protein is already at a highlevel. The effect of toxic substances can be shown more effectively whena strong and fast-induced promoter is used to regulate a certain gene oraction in a rec-DNA vector.

The Bacterial Strains, Plasmids and their Construction, Methods used inthe Invention

As cloning hosts and in toxicity measurements E. coli JM 103 (lac-pro,thi, strA, supE, enda, sbsB15, hsdR4 (F'traD34, proAB, lacI^(q)Z M15)(Messing et al., Nucl. Acids Res., 9, 1981, 309-321), MC1061 (cI⁺,araD139, (ara-leu)7696, lacX74, galU⁻, galK⁻, hsr⁻, hsm⁺, strA)(Casadaban & Cohen, J. Mol. Biol., 138, 1980, 179-207), BW322 (CGSC,rfa-210: :Tn10, thi-1, relAl, spoT1, pyrE) and K-12 (M72 Sm^(R)-lacZambio-uvrB, trpEA2 (Nam7Nam53cI857 HI) (Remaut et al., 1981, GENE, 15,81-93) and Bacillus subtilis 1A40 (Bacillus Genetic Stock Center, lys-3,metB10, trpC2) were used. Cells were grown on appropriate minimal agarplates and were kept maximally one month at +4° C. after which newplates were streaked. The strains were also kept in 15% glycerol at −70°C., wherefrom growth was initiated on minimal plates. Cells for plasmidextraction were first cultivated in 5 ml of 2×TY medium (16 g Bactotryptone, 8 g Yeast extract, 8 g NaCl, H₂0 to 1 l, pH 7.4, withappropriate antibiotic) 10 h at 30° C. in a shaker after which theculture was transferred to a larger volume for 10 h in the same medium.

FIGS. 5a and 5 b shows the construction of a rec-DNA plasmid pCSS123(deposited with Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (DSM), Mascheroder Weg 1 B, D-3300 Braunschweig, under the BudapestTreaty on Jan. 2, 1989, with a DSM number 5119), in FIG. 5c theconstruct of a rec-DNA plasmid pCSS302 deposited with DSM under theBudapest Treaty on Mar. 3, 1993, with a DSM number of 7503, and in FIG.5d the construct of a rec-DNA plasmid pCSS305 deposited with DSM underthe Budapest Treaty on Mar. 3, 1993, with a DSM number of 7504. PlasmidpWH102 (Gupta et al., 1985, Arch. Microbiol., 143, 325-329) was cut withthe restriction enzymes SalI and PvuII and fragments were separated byagarose gel electrophoresis. A DNA band of 2300 base pairs (bp) was cutout of the gel under UV light. The low-gelling temperature agarose wasmelted at 65° C. and the DNA band was ligated with DNA ligase to plasmidpEMBL19(−) (Dente et al., 1983, Nucleic Acids Res., 11, 1645-1655) cutwith SalI and SmaI. The plasmid obtained was transformed into E. coliJM103 cells using a method described below. A plasmid extraction inmini-scale was performed according to Maniatis et al. (1982, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor) and the correct constructions were verified with suitablerestriction enzyme analysis. Plasmid extraction in large scale wasperformed according to the same manual. The plasmid shown in FIG. 5a(pCSSlll) was cut with the restriction endonuclease PvuII and aDNA-piece of 2400 bp was separated as described earlier. This piece,containing bacterial luciferase genes from V. harveyi under lac promotercontrol, and was ligated to plasmid pOU61 (Larsen et al., 1984, GENE,28, 45-54) which was cut with the restriction enzyme BamHI and filled inwith Klenow DNA-polymerase enzyme. Ligation mixture was transformed toE. coli JM101 strain as described below and correct plasmid containingcolonies were picked by their ability to produce light after visualchecking of the plates in a dark room. This was performed by adding 5 μlof 10% decanal on the lid of cultivation plate, which revealed the lightproducing colonies within a few minutes after the aldehyde hadpenetrated the cells. The plasmid obtained is shown in FIG. 5b and wasnamed pCSS123. An analogous runaway-replication plasmid pCSS302 wasconstructed as follows: plasmid pLucGR(tac) (Wood et al., 1989, Science,244, 700-702) was cut with the restriction endonucleases XhoI andHindIII and filled in with Klenow enzyme. After separation of thefragments in an agarose gel, a 1800 bp fragment containing the geneencoding green luciferase of click beetle under the control of tacpromoter was ligated to plasmid pOU61 which was cut with BamHI andfilled in as described above. Ligation mixture was transformed in E.coli JM103 and correct transformants were verified from plasmidminipreparations and the resulting plasmid pCSS302 is shown in FIG. 5c.A second analogous runaway-replication plasmid pCSS305 was constructedas follows: plasmid pCGLSll (K. Nealson, personal communication and inpress) was digested with the restriction enzyme PvuII and a 7 kbfragment containing the genes encoding luciferases α- and β-subunits ofX. luminescens under the control of lac promoter of E. coli were ligatedto plasmid pOU61 which was cut with BamHI and filled in as describedabove. Ligation mixture was transformed in E. coli JM103 and correcttransformants were verified from plasmid minipreparations.

The symbols and abbreviations used: amp^(r)=gene encoding β-lactamase,ori=the origin of replication of the plasmid, luxA and B=genes encodingthe subunits of luciferase, lacp=the promoter of the E. coli lactoseoperon, F1(IG)=the intergenic region of phage F1, MCS=multible cloningsite of pUC18 (Yanisch-Perron et al., 1985, GENE, 33, 103109),kb=thousand base pairs, repA, copA and cobB=genes encoding proteinsresponsible for plasmid copy number and partitioning of plasmid intodaughter cells, and cI857=the temperature sensitive repressor of phagelambda. The abbreviations of restriction endonucleases used: R=EcoRI,H=Hindiii, B=BamHI, S=SalI, P=PvuII, Sa=SacI, K=KpnI, Sm=SmaI, X=XbaI,Ps =PstI, Sp =SphI. B/P=the ligation point BamHI, filled in, PvuII.B/H=ligation point BamHI, filled in, HindIII. B/Xh=ligation point BamHI,filled in, XhoI.

FIG. 6 shows plasmid pCSS108 (Korpela & Karp, Biotechnol. Lett., 10,1988, 383-388), which is used for production of bacterial luciferase byadding a chemical called IPTG. IPTG triggers protein production bybinding to lac repressor protein. The genes encoding luciferase fromVibrio harveyi were transferred from the plasmid pWH102 (Gupta et al.,1985, Arch. Microbiol., 143, 325-329) by cutting the plasmid with therestriction enzyme BamHI. The two pieces obtained were treated withenzyme alkaline phosphatase (CIP) to remove the terminal phosphategroups so that the pieces can not ligate to themselves. A piece of 5000bp was separated in an agarose gel as described previously. Thisfragment was ligated using T4-DNA ligase to a plasmid pEMBL18(+) whichhad been cut with the same enzyme. After transformation to E. coli JM103and overnight incubation of the transformants, the cultivation plateswere screened for light producing colonies as described above. Symbolsused are as in FIG. 5.

The construction of plasmid pCSS112 (deposited as a DSM number 5120) isshown in FIG. 7a. The plasmid contains luciferase genes from V. harveyiand the genes are under the control of the P_(L) promoter of phagelambda. The promoter is regulated by the repressor protein cI857 ofphage lambda. When cultured in a suitable bacterial host such as E. coliK-12 HI trp, the repressor protein can be destroyed by heat treatment.Plasmid pWH102 was cut with restriction enzymes SalI and BamHI. PlasmidpPLcAT110 (Stanssens et al, 1985, GENE, 36, 211-233, partly unpublished)was cut with restriction enzymes SalI and BglII. The DNA fragments wereseparated as described earlier and a 3200 bp piece from plasmid pWH102and a 2900 bp piece of pPLcAT110 were ligated with the aid of T4-DNAligase. After transformation into E. coli MC1061 (cI⁺) the correctplasmid containing transformant was screened as described above. Theplasmid obtained was transformed to E. coli K-12 HI trp host. FIG. 7bshows a plasmid pCSS301 which is basically similar to pCSS112 except agene encoding green click beetle luciferase (Wood et al., 1989, Science,244, 700-702) is present. A plasmid pLucGR(tac) containing theluciferase gene from click beetle was digested with restriction enzymeBspHI, the cohesive ends were made blunt by Mung bean nuclease treatmentand a DNA fragment of 1643 bp was separated in an agarose gel asdescribed. This fragment was ligated to XbaI-BglII—digested vectorpPLcAT110 (described earlier), which was filled in and CIP-treated. Theligation mixture was first transformed to E. coli MC1061 cells and afterthe correct plasmid was found from plasmid minipreparations it wastransformed to E. coli K-12 HI trp. The symbols used are as in FIG. 5,also P_(L)=leftward promoter of phage lambda., Pv=PvuI, Xb/Bs=ligationpoint XbaI, filled in, BsphI, Mung bean treated, Bs/Bg=ligation pointBsphI, Mung bean treated, BglII, filled in.

Plasmid pCSS962 was constructed as follows: A shuttle vector p602/22,which can replicate both in E. coli and in B. subtilis (LeGrice et al.,1987, GENE, 55, 95-103) was cut with restriction enzyme BamHI, filled inwith Klenow enzyme and treated with calf intestinal alkaline phosphatase(CIP). The plasmid pLucGR(tac) containing a green click beetleluciferase gene was digested with restriction enzyme BspHI, filled inwith Klenow enzyme and separated on an agarose gel as described above.The gene and the vector were ligated together. The ligation mixture wastransformed into E. coli MC1061 as described below and correct plasmidconstructions were verified by analyzing the plasmid minipreparationswith suitable restriction enzyme analysis. The correct plasmid wasco-transformed with a helper plasmid pBLl (LeGrice et al., 1987, GENE,55, 95-103) into B. subtilis 1A40 strain as described below. The plasmidand its construction are shown in FIG. 7c. The symbols used are:P/O=Promoter/Operator; ori+=E. coli origin of replication; ori+=B.subtilis origin of replication; kan=gene encoding kanamycinacetyltransferase; cat=gene encoding chloramphenicol acetyltransferase;T1=transcriptional terminator.

The competence induction of E. coli strains: E. coli strains were madecompetent, i.e., able to take foreign DNA inside the cell as follows: E.coli were grown overnight in a volume of 5 ml in 2×TY medium andtransferred to 100 ml of the same medium. After about two hours, theoptical density, as measured at 600 nm, was 0.8. The cells were cooledin an ice bath and centrifuged at 4000×g for 5 min. The cell pellet wassuspended in 50 ml of 50 mM CaCl₂ and centrifuged at 3000×g for 5 min at0° C. The cells were suspended in 4 ml of 50 mM CaCl₂ containingglycerol 15%. These competent cells were divided in 1 ml aliquotes andthey were frozen rapidly in liquid nitrogen and stored at −70° C. forlater use.

The competence induction of B. subtilis: B. subtilis 1A40 was grownovernight in 5 ml of 2×TY at 37° C., spun down and suspended in 15 ml ofGrowth Medium 1 [(SMS=(NH₄)₂SO₄ 0.2%, K₂HPO₄ 1.4%, KH₂PO₄ 0.6%,Na-citrate 0.1%, MgSO₄ 0.02%), glucose 0.5%, casamino acids 0.05%, yeastextract 0.06%, MgCl₂ 1.5 mM] and grown until the optical density was 1.8as measured at 600 nm. The culture was then transferred to 150 ml ofGrowth Medium 2 [(SMS, glucose 0.5%, casamino acids 0.01%, yeast extract0.025%, MgCl₂ 5 mM, Ca(NO₃)₂) 2.5 mM] for 90 minutes at 37° C. Aftercentrifugation at 8000 rpm for 5 minutes at room temperature, the pelletwas suspended in 15 ml of the supernatant. Glycerol was added to 8% andcells were divided into 1 ml aliquotes and quickly frozen in liquid N₂and stored at −70° C.

The Transformation of E. coli Strains with rec-DNA Plasmids

One to ten μl plasmid-DNA or ligation mixture was added tomicrocentrifuge tubes prechilled in an ice bath. To these, 250 μl ofcompetent-cells and 26 μl of 10×TMC (100 mM TRIS-HCl, pH 7.4, 100 mMMgCl₂, 100 mM CaCl₂) were added and kept in an ice bath for 10 min withoccasional careful mixing. The cells were heat shocked for two minutesat 42° C. and then one ml of 2×TY was added. The cells were keptthereafter at 30° C. for one hour and centrifuged for 3 min at 8000×g.The supernatant was discarded and cells were suspended in the leftoversupernatant (about 100 μl). The cell suspension was spread on antibioticselection plates and incubated at 30° C. overnight.

The transformation of B. subtilis with rec-DNA plasmids: One ml offrozen competent B. subtilis cells were quickly melted in a 37° C.waterbath and they were diluted in 10 ml of SMS Dilution Medium (SMS,glucose 0.5%, MgCl₂ 20 mM, EDTA 1 mM). One ml of diluted cells weremixed with 1 μg of pCSS962 and 1 pg of pBL1 and incubated at 37° C. for30 minutes with shaking. Subsequently, cells were plated on 2×TY platescontaining 10 μg/ml of kanamycin and erythromycin. Plates were incubatedat 30° C. for 22 hours.

Example 1 The Change in Plasmid Copy-number when Cells are Treated withNalidixic Acid

The plasmid pCSS123 described in the instant invention is arunaway-replication plasmid, in which a change in copy number can beobtained by shifting the temperature (FIG. 8). The effect of nalidixicacid, an agent known to inhibit DNA replication of cell DNA andespecially of plasmid pCSS123 DNA, is shown in FIG. 8. FIG. 8 shows theamount and quality of plasmid-DNA extracted from heat-treated E. colicells.

E. coli pCSS123/JM103 cells were cultivated in 20 ml of 2×TY at 30° C.in four Erlenmeyer bottles until the absorbance measured at 600 nm was0.3. Nalidixic acid, a known inhibitor of DNA replication, was added toa final concentration of 0, 1, 10 and 100 μg/ml. Parallel samples of 1.5ml from each bottle were immediately withdrawn to 15 ml tubes and keptat 30° C. for 20 min. The tubes were transferred to 42° C. for one hourand the bottles were left at 30° C. After one hour, both the tubes andthe bottles were kept for an additional hour at 30° C. in a shaker.Total-DNA was extracted from 1.5 ml of culture.

Cells were centrifuged and the pellets were suspended in 500 μl 50 mMTRIS-HC1, pH 8.0, 50 mM EDTA. Cells were kept in an ice bath for 30 minand 50 μl of lysozyme (10 mg/ml) was added and kept in an ice bath for45 min. One hundred μl of STEP solution (0.5% SDS, 50 mM TRIS-HC1, pH7.5, 0.4 M EDTA) was added and kept at 50° C. for 60 min. After this,600 μl of phenol was added and the tubes were gently mixed for 5 min andcentrifuged 10 min at 12000×g. Two volumes of absolute ethanol andK-acetate, pH 6.0 to 0.3 M, were added to the supernatant to precipitatethe DNA. After 30 min at −70° C. the tubes were centrifuged 10 min at12000×g and the pellet was washed with 500 μl of 70% ethanol,centrifuged and the pellets were dried in a vacuum exiccator for 5 min.The dried pellet was dissolved in 50 μl of 50 mM TRIS-HC1, pH 7.5, 1 mMEDTA, 100 μg/ml RNAase A solution and kept at 65° C. for 20 min. Aconventional agarose gel electrophoresis analysis was made for theextracted DNA's. As shown in FIG. 8, the DNA content did not change inthe samples which were not heat-treated. In contrast, the DNA contentincreased several fold in samples which were heat-treated and nottreated with nalidixic acid. DNA content was unaffected by the presenceof 1 μg or higher nalidixic acid whether the cells were heat-treated ornot.

Example 2 Determination of Toxic Substances as Measured by the Help ofLight Production by Cells Containing Plasmid Whose Copy Number Can beChanged

E. coli pCSS123/JM103 cells grown overnight were diluted 1:1000. Dilutedcells in 2×TY were taken (0.5 ml) and various amounts of antibiotics orother toxic substances were added. These solutions were incubated 20 minat 30° C. After this the samples were transferred to 42° C. for onehour. Each tube was thereafter incubated at 30° C. in a waterbath andIPTG was added to 1 mM and n-decanal to 0.01%. The tubes weretransferred to an automated light-gathering device, i.e., luminometer1251 (LKB-Wallac, Turku, Finland) whose measuring chamber had beenequilibrated to 30° C. Measurement of light emission by the cells wasdone with the auto-mode program so that each tube was automaticallymeasured every two minutes. Data were collected in the memory of thecomputer for later analyses. FIG. 9a shows the detection of nalidixicacid using E. coli cells cloned with pCSS123 As shown in FIG. 9a, 2 μgof nalidixic acid can be detected very quickly. FIG. 9b shows thedetection of chloramphenicol using E. coli cells cloned with pCSS123.For clarity reasons only two concentrations of chloramphenicol werecompared to the untreated control. FIG. 9c shows the detection of heavymetal cadmium using E. coli cells cloned with pCSS123.

Freeze-dried E. coli pCSS123/BW322 were reconstituted with 1.0 ml of2×TY and 45 μl of this was diluted 1:10 with 2×TY. Five μl oftrimethoprim dilutions were added and kept at room temperature for 25minutes. Cells were heat induced at 42° C. for 25 minutes andequilibrated to 30° C. in a water bath for 10 minutes. n-decanal wasadded to 0.001% and light production was measured using a LKB-Wallac1250 manual luminometer. Same concentrations of trimethoprim togetherwith reconstituted, freeze-dried cells which were not heat-treated actedas controls. FIG. 9d shows the detection of trimethoprim using E. colicells cloned with pCSS123. As shown in FIG. 9d, the sensitivity of theassay is very high. FIG. 9e shows the detection of oflaxacin using thesame approach as in FIG. 9d.

One hundred μl of reconstituted E. coli pCSS123/BW322 were plated onto aPetri dish. The effect of UV light (254 nm) was tested by varying thetime of exposure. Following exposure, the samples were transferred to42° C. for 45 min and n-decanal to 0.01% was added. Light output wasdetermined as described above. FIG. 9f shows light output for E. colicells cloned with pCSS123 and treated with UV light.

E. coli pCSS302/BW322 cells grown overnight were diluted in 2×TY. Ninetyμl of the diluted cells were taken and antibiotics (laxacin orcitrofloxacin) were added. The solutions were incubated for 20 min atRT. After this, the samples were transferred to 42° C. for 45 min. Thecells were measured for light production by adding 100 μl of solutioncontaining 1 MM D-luciferin and 1 mM ATP in 0.1M Na-citrate buffer, pH5.0. FIG. 9g the detection of oflaxacin using E. coli cells cloned withpCSS302. FIG. 9h shows the detection of citrofloxacin using E. colicells cloned with pCSS302.

E. coli pCSS305/BW322 cells were used to test a system where nosubstrate addition was needed to produce light from cells. To 90 μl ofthe cells in 2×TY, 10 μl of the antibiotics was added. Differentconcentrations of oflaxacin (FIG. 9j) and citrofloxacin (FIG. 9i) wereadded and the tubes were kept at RT for 25 min. After this the inductionwas done by shifting the tubes to 42° C. for 45 min. The tubes wereloaded in the luminometer for light production measurement. FIG. 9jshows the detection of oflaxacin using E. coli cells cloned withpCSS305. FIG. 9i shows the detection of citrofloxacin using E. colicells cloned with pCSS305.

Example 3 The Detection of Antibiotics with a Method Where Control ofPlasmid Replication is not Possible

A comparison is made between plasmids where the expression of bacterialluciferase genes are controlled by either lac (slow) or P_(L) promoter(fast) promoters. E. coli clone pCSS112/K-12 HI trp(c1857) was grownovernight in 2×TY medium containing ampicillin 100 μg/ml. After this asuitable dilution was made in HBSS-buffer or in milk and 500 μl of thiswas added to 3 ml luminometer tubes. The tubes were equilibrated to 30°C. and different amounts of various toxic substances were added totubes. Tubes were kept at 30° C. for 20 min, after which the temperaturewas shifted to 42° C. for 10 minutes. Thereafter the tubes were removedto luminometer chamber which had been equilibrated to 30° C. forautomated measurement. As a comparison, an E. coli JM103 clonecontaining plasmid pCSS108 was used as a control. The control cells weretreated similarly but without the heat-shock step. Messanger RNA andprotein synthesis were induced in the control cells by adding IPTG to 1mM. FIG. 10 shows a comparison of the detection of toxic substances inE. coli cells where protein synthesis is directed by the P_(L) promoteror the lac promoter. As shown in FIG. 10, when P_(L) promoter directsprotein synthesis it is possible to detect toxic substance in much lowerconcentrations than using the slower and weaker lac promoter. In case ofchloramphenicol, the kinetics of light production have been shown inFIG. 11a when plasmid pCSS112 in E. coli K-12 HI trp(c1857) strain isused. FIG. 12 shows that differences in the measured activity (lightproduction) are seen from the start of measurement even withconcentrations as low as 0.1 μg/ml in the measuring cuvette.

The detection of antibiotics belonging to the penicillin family is ofutmost importance since these antibiotics are very widely used and quickmethods do not exist to detect their presence. FIG. 11b shows thedetection of ampicillin, oxytetracyclin, and streptomycin using B.subtilis 1A40 cells cloned with luciferase gene from a click beetle. Theplasmid used is shown in FIG. 7c. Using this construction, theproduction of luciferase can be turned on in B. subtilis by simpleaddition of IPTG, which binds to the lac repressor coded by the helperplasmid pBL1 present in the same cell. After IPTG binds to therepressor, the repressor cannot bind to the DNA region between phage T5promoter and the luciferase gene thus allowing the expression ofluciferase. Cells containing both plasmids were cultured overnight in2×TY containing erythromycin (10 μg/ml, to keep pBL1 in the cell) andkanamycin (10 μg/ml, to keep pCSS952 in the cell). A suitable dilutionwas made and different amounts of antibiotic were added to the cells.After an incubation period of 2 hours at 30° C., the tubes were measuredfor light emission after addition of 1 mM D-luciferin substrate in 0.1 MNa-citrate. As shown in FIG. 11b a low amount of 0.1 ug/ml ampicillinand even lower amounts of oxytetracyclin and streptomycin can bedetected.

Example 4 Detection of Toxic Substances Using a Method Where E. coliContains Constant Copy-number rec-DNA Plasmid and in Which Promoter ofPhage Lambda Controls the Biosynthesis of Bacterial Luciferase and ClickBeetle Luciferase

The following examples show detection of substances affecting otherbiosynthetic routes and metabolism of cells. The tests have beenperformed in the same way as those described in previous examples. Thegoal has been to develop an extremely rapid method, which is also verysensitive. Plasmid pCSS112 cloned in the E. coli K-12 strain was usedthroughout the following examples.

The following figures show the effect of each tested substance asmeasured by light production. As in earlier measurements, the presenceof inhibiting factors is seen as lowered light production compared tocases where the factor has not been present. Shown in FIG. 12 is thedetection of an antibiotic, rifampicin, which is a known inhibitor oftranscription, i.e., formation of messenger RNA.

Oxytetracycline is an antibiotic which binds to the 30S ribosomalsubunit. FIG. 13 shows the effect of oxytetracycline on light output inE. coli cells cloned with pCSS112. Amounts as low as one μg can be seenvery rapidly with the method described in this invention. The effect ofoxytetracycline is strong and easily detected. The effect of sulphitewhich is a known inhibitor of metabolism and used in food processing isshown in FIG. 14a. The effect of heavy metal cadmium, which is also aknown inhibitor of metabolism and contaminates soil and water, is shownin FIG. 14b. These results show that the test system described in thisinvention is also applicable to the quick determination of metabolicinhibitors. In addition, it shows that the method can detect thepresence of agents other than antibiotics.

In addition to bacterial luciferase, other luciferases can be usedwithout losing the sensitivity or performance of the test. FIG. 15 showslight output of E. coli cloned with pCSS301 and treated withoxytetracycline or rifampicin The plasmid (pCSS301) used in this testcontains the click beetle luciferase gene as described in FIG. 7b. Thetest was done essentially as that described for bacterial luciferaseexcept that after the cells were incubated with or without toxicsubstances, the cells were measured for light production after 15minutes at 30° C. by adding 100 μl of solution containing 1 mMD-luciferin, 1 mM ATP in 0.1 M Na-citrate buffer, pH 5.0. Thereafter thelight production was measured using a manual luminometer 1250(LKB-Wallac, Turku, Finland). As can be seen from FIG. 15, thesensitivity of the method to detect either oxytetracycline or rifampicinis extremely high and comparable to the detection made with bacterialluciferase.

Example 5 The Determination of a Toxic Substance Using a Method WhereP_(L—) promoter of Phage Lambda Activates the Biosynthetic Machinery toProduce β-galactosidase

In the previous examples, measurements were based on light produced fromluciferase genes. However, any protein or peptide can be used provided amethod for detecting the protein or peptide exists. In this example,β-galactosidase is used in place of luciferase. The plasmid pPLcAT14used has been described elsewhere (Stanssens, P., Remaut, E. & Fiers,W., 1985, GENE, 36, 211-223).

E. coli clones pPLcAT14/K-12 HI trp were grown overnight in 2×TY mediumwhich was supplemented with ampicillin 100 μg/ml. After this a suitabledilution was made from bacteria in HBSS buffer and 80 μl of this wasadded to glass tubes. Different concentrations of chloramphenicol wereadded and tubes were kept at room temperature for 15 minutes. Afterincubation, activation of the P_(L) promoter was induced by shifting thetubes to 42° C. for 30 minutes. As a consequence the biosynthesismachinery is activated to produce β-galactosidase encoded by theβ-galactosidase gene cloned under P_(L) promoter in plasmid pPLcAT14.After induction, toluene was added to 10%. Toluene makes the cellsporous to a chemical, ONPG. Following reaction of ONPG withβ-galactosidase, a yellow color forms. The yellow color can be measuredwith a spectrophotometer at 420 nm. FIG. 16 shows the amount ofβ-galactosidase produced in E. coli cloned with pPLcAT14, induced withIPTG in the presence of chloramphenicol.

Example 6 Determination of Organic Content in a Solution Using a MethodWhere P_(L) promoter of Phage Lambda Activates the BiosyntheticMachinery to Produce Luciferase

E. coli pCSS112/K-12 HI trp cells were cultivated overnight in 2×TYcontaining ampicillin (100 μg/ml). Cells were spun down and washed twicewith a HBSS medium (Korpela & Karp, 1988, Biotech. Lett., 10, 383-388)omitting glucose and gelatine but supplemented with tryptophane 0.02%.The cells were shaken in this medium for 4 hours, spun down andsuspended in HBSS buffer containing either 0.1% glucose or 0.1%(NH₄)₂SO₄ depending on whether carbon sources or nitrogen sources wereevaluated, respectively. A suitable dilution was made from treated cellsin minimal salts and various amounts of either carbon or nitrogensources were added. The cells were incubated 10 minutes at 30° C. A heattreatment of 10 minutes at 42° C. was given to the cells to start theprotein synthesis. The cells were then incubated 10 minutes at 30° C.and then the tubes were loaded in the automated luminometer for lightproduction measurements after addition of n-decanal to 0.001%. FIG. 17shows the detection of glucose.

TABLE 1 Biochemical targets for drug action: Inhibitors of Inhibitors ofprotein nucleic acid Cell walls synthesis synthesis beta-lactamschloramphenicol nalidixic acid cephalosporins tetracyclines novobiocinbacitracin aminoglycosides rifamycins vancomycin macrolides phleomycinpolymyxins erythromycin mithramycin gramicidins lincomycin actinomycinvalinomycin puromycin quinolones

What is claimed is:
 1. A method for determining the presence or amountof at least one inhibitory factor in the sample to be tested, whereinthe inhibitory factor inhibits one or more of DNA synthesis,transcription of DNA, translation of RNA, cell wall synthesis, cellmembrane function or metabolic functions which participate in orinfluence these processes, said method comprising: a) incubating asample to be tested with a population of transformed cells for a periodsufficient to allow an inhibitory factor, if present in the sample, toaffect said cells, said cells being transformed with a recombinant DNAplasmid, and replication of said plasmid being inducible by an exogenousstimulus independent of replication of the cells and further whereinsaid cells are sensitive to inhibition by the inhibitory factor; b) thenapplying said exogenous stimulus to said cells in an amount sufficientto induce replication of said plasmid in the absence of the inhibitoryfactor; and c) determining the amount of DNA produced by said populationof cells, the amount of the inhibitory factor in said sample beingcorrelated with a reduction in the amount of DNA produced compared tothe amount of DNA produced by a like cell population by application of alike amount of exogenous stimulus in the absence of said sample.
 2. Themethod of claim 1, wherein said exogenous stimulus is change oftemperature.
 3. The method of claim 1, wherein said exogenous stimulusis a chemical inducer.
 4. The method of claim 1, wherein saidrecombinant plasmid contains DNA encoding an expressible enzyme and saidstep of determining the amount of DNA comprises measuring the activityof said enzyme.
 5. A method according to claim 1, wherein therecombinant DNA plasmid contains a DNA sequence which encodes at leastone selected protein or a part of said selected protein that isessential for biological activity of said protein and wherein therecombinant DNA plasmid comprises one or more DNA sequences which makethe cell resistant to an antibiotic, heavy metal or toxin.
 6. A methodaccording to claim 5, wherein the DNA sequence encoding the protein issubject to a regulatable promoter, which is controlled by positive ornegative feedback and is regulatable in said cells and is activatedsimultaneously or at a desired moment subsequent to induction of plasmidreplication.
 7. A method according to claim 6, wherein the factor isselected from the group consisting of aflatoxins, heavy metals, ethidiumbromide, nalidixic acid, trimethoprim, fluoroquinolones,aminoglycocides, penicillines, cephalosporines, rifampicin,chloramphenicol, tetracyclines, and sulphonamides, and wherein the cellused is sensitive to said factor.
 8. A method according to claim 7,wherein the cells are Escherichia coli and replication of therecombinant DNA plasmid contained in the cells can be accuratelyregulated by means of a strong promoter selected from the groupconsisting of lambda P_(L) and P_(R) promoters, lac, trp, and hybrid tacpromoters.
 9. A method according to claim 8, characterized in that therecombinant DNA plasmid is pCSS 123, pCSS302 or pCSS305, deposited underDSM number 5119, 7503 or 7504, respectively.
 10. A method fordetermining the presence or amount of at least one inhibitory factor inthe sample to be tested, wherein the inhibitory factor inhibits one ofmore of DNA synthesis, transcription of DNA, translation of RNA, cellwall synthesis, cell membrane function or metabolic functions whichparticipate in or influence these processes, said method comprising: a)incubating a sample to be tested with a population of transformed cellsfor a period sufficient to allow the inhibitory factor, if present inthe sample, to affect said cells, said cells being transformed with ahigh copy number recombinant DNA plasmid containing a sequence encodinga marker protein which is detectable when expressed, said sequence beingcoupled to a regulatable promoter such that expression of said markerprotein is inducible by an exogenous stimulus; b) then applying saidexogenous stimulus to said population of cells in an amount sufficientto induce expression of said marker protein in the absence of theinhibitory factor; and c) determining the amount of said marker proteinexpressed by said population of cells, the amount of the inhibitoryfactor in the sample being correlated with a reduction in the amount ofsaid marker protein produced compared to the amount of said markerprotein produced by a like cell population by application of a likeamount of exogenous stimulus in the absence of the sample.
 11. Themethod of claim 10, wherein said marker protein is luciferase.
 12. Amethod according to claims 1 or 10, wherein the cell is a gram negativeor gram positive bacteria belonging to the group Enterobacteriaceae orthe group Bacillus.
 13. A method according to claims 1 or 10, whereinthe recombinant DNA plasmid comprises one or more DNA sequences, whichmake the cell resistant to an antibiotic, heavy metal, or toxin.
 14. Amethod according to claims 1 or 10, wherein the sample to be tested isan aerosol.
 15. A method according to claims 1 or 10, wherein thepopulation of cells is lyophilized and is rehydrated before the step ofincubating by a suitable liquid or cultivation medium.
 16. A methodaccording to claims 1 or 10, wherein the factor is selected from thegroup consisting of mutagens, antibiotics, heavy metals and toxins. 17.A method according to claim 16, wherein said cells are Bacillussubtilis.
 18. A method according to claim 16, wherein said cells areEscherichia coli.
 19. A method according to claim 17, wherein the markerprotein is subject to a regulatable strong promoter selected from thegroup consisting of φ 105, phage T5 promoter controlled by lac operatorand saccharose regulatable promoter, and the marker protein is selectedfrom the group consisting of alpha-amylase, alkaline phosphatase,β-galactosidase, luciferase, peroxidase, T4 lysozyme, β-glucuronidase,oxidoreductase and pyrophosphatase.
 20. A method according to claim 18,wherein the recombinant DNA plasmid contains a DNA sequence that encodesa marker protein and the expression of said protein is controllable bymeans of a regulatable promoter, selected from the group consisting oflac, trp, lambda P_(R) and P_(L), and tac promoters.
 21. A methodaccording to claim 20, wherein the protein is selected from the groupconsisting of luciferase, β-galactosidase, alkaline phosphatase,peroxidase, T4 lysozyme, β-glucuronidase, oxidoreductase andpyrophosphatase.
 22. A method according to claims 19 or 21, wherein therecombinant DNA plasmid encodes a luciferase enzyme.
 23. A methodaccording to claim 22, wherein the recombinant DNA plasmid is plasmidpCSS112, pCSS301 or pCSS962.
 24. A method according to claim 22, whereinan aldehyde is used to determine the amount of expressed luciferase insaid cells.
 25. A method according to claims 1 or 10, wherein the sampleto be tested is milk, serum or water.
 26. The method of claim 4, whereinsaid enzyme is luciferase.
 27. A method for determining the presence ofat least one inhibitory factor selected from the group consisting ofampicillin, chloramphenicol, oxytetracycline, streptomycin,erythromycin, ofloxacin, ciprofloxacin, actinomycin and trimethoprim ina sample of a biological fluid selected from the group consisting ofblood, plasma, serum, urine, semen and milk, said method comprising: a)obtaining a population of cells transformed with a high copy numberrecombinant DNA plasmid containing a sequence encoding luciferase,wherein said sequence is coupled to a regulatable promoter such thatexpression of said sequence is inducible by application of a chemicalinducer to cells containing said plasmid; b) incubating said sample withsaid cell population in a medium suitable for growth of said cells for aperiod sufficient to allow said inhibitory factor if present to affectsaid cells; c) then applying said chemical inducer to said cells in anamount which in the absence of said inhibitory factor is sufficient toinduce expression of said sequence; and d) determining the amount ofluciferase expressed by said cell population, the amount of saidinhibitory factor in said sample being correlated with the reduction inthe amount of luciferase produced relative to the amount of luciferaseproduced by a like cell population by application of a like amount ofsaid chemical inducer in the absence of said sample.
 28. A methodaccording to claims 1 or 10 wherein the sample to be tested is a bodyfluid of an animal, soil, water or milk.